Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite

Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-( S )-sulphoxide, and MsrB, active against methionine-( R )-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects Δ msrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N -acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.     

Identification of substrates of the Mycobacterium tuberculosis proteasome

The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice.     

PUPylation provides the punch as Mycobacterium tuberculosis battles the host macrophage

The proteasome machinery has been shown to provide Mycobacterium tuberculosis (Mtb) with the ability to protect itself from the damaging effects of reactive nitrogen intermediates. In their recent paper, Darwin and colleagues identify the protein modifier in Mtb that targets substrates for degradation in the Mtb proteasome.     

Virulence of Mycobacterium tuberculosis depends on lipoamide dehydrogenase, a member of three multienzyme complexes

Mycobacterium tuberculosis (Mtb) adapts to persist in a nutritionally limited macrophage compartment. Lipoamide dehydrogenase (Lpd), the third enzyme (E3) in Mtb's pyruvate dehydrogenase complex (PDH), also serves as E1 of peroxynitrite reductase/peroxidase (PNR/P), which helps Mtb resist host-reactive nitrogen intermediates. In contrast to Mtb lacking dihydrolipoamide acyltransferase (DlaT), the E2 of PDH and PNR/P, Lpd-deficient Mtb is severely attenuated in wild-type and immunodeficient mice. This suggests that Lpd has a function that DlaT does not share. When DlaT is absent, Mtb upregulates an Lpd-dependent branched-chain keto acid dehydrogenase (BCKADH) encoded by pdhA, pdhB, pdhC, and lpdC. Without Lpd, Mtb cannot metabolize branched-chain amino acids and potentially toxic branched-chain intermediates accumulate. Mtb deficient in both DlaT and PdhC phenocopies Lpd-deficient Mtb. Thus, Mtb critically requires BCKADH along with PDH and PNR/P for pathogenesis. These findings position Lpd as a potential target for anti-infectives against Mtb.     

Selective killing of nonreplicating mycobacteria

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.     

Important role for Mycobacterium tuberculosis UvrD1 in pathogenesis and persistence apart from its function in nucleotide excision repair

Mycobacterium tuberculosis survives and replicates in macrophages, where it is exposed to reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the roles of UvrA and UvrD1, thought to be parts of the nucleotide excision repair pathway of M. tuberculosis. Strains in which uvrD1 was inactivated either alone or in conjunction with uvrA were constructed. Inactivation of uvrD1 resulted in a small colony phenotype, although growth in liquid culture was not significantly affected. The sensitivity of the mutant strains to UV irradiation and to mitomycin C highlighted the importance of the targeted genes for nucleotide excision repair. The mutant strains all exhibited heightened susceptibility to representatives of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). The uvrD1 and the uvrA uvrD1 mutants showed decreased intracellular multiplication following infection of macrophages. Most importantly, the uvrA uvrD1 mutant was markedly attenuated following infection of mice by either the aerosol or the intravenous route.     

A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis

Acidification of the phagosome is considered to be a major mechanism used by macrophages against bacteria, including Mycobacterium tuberculosis (Mtb). Mtb blocks phagosome acidification, but interferon-gamma (IFN-gamma) restores acidification and confers antimycobacterial activity. Nonetheless, it remains unclear whether acid kills Mtb, whether the intrabacterial pH of any pathogen falls when it is in the phagosome and whether acid resistance is required for mycobacterial virulence. In vitro at pH 4.5, Mtb survived in a simple buffer and maintained intrabacterial pH. Therefore, Mtb resists phagolysosomal concentrations of acid. Mtb also maintained its intrabacterial pH and survived when phagocytosed by IFN-gamma-activated macrophages. We used transposon mutagenesis to identify genes responsible for Mtb's acid resistance. A strain disrupted in Rv3671c, a previously uncharacterized gene encoding a membrane-associated protein, was sensitive to acid and failed to maintain intrabacterial pH in acid in vitro and in activated macrophages. Growth of the mutant was also severely attenuated in mice. Thus, Mtb is able to resist acid, owing in large part to Rv3671c, and this resistance is essential for virulence. Disruption of Mtb's acid resistance and intrabacterial pH maintenance systems is an attractive target for chemotherapy.     

Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis

The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.     

The production of a 70 kDa heat shock protein by Toxoplasma gondii RH strain in immunocompromised mice

A 70 kDa heat shock protein (HSP70) has been reported previously to be strongly expressed in virulent Toxoplasma gondii strains taken from immunocompetent mice but it is poorly expressed by virulent parasites in mice immunocompromised by treatment with cortisone acetate or by virulent parasites cultured in vitro. Immune factors such as interferon-γ, tumour necrosis factor and reactive nitrogen intermediates derived from nitric oxide are known to be important inducers of HSP70 production and are also known to be produced during the immune response to acute T. gondii infection. The ability of these immune factors to induce T. gondii HSP70 production was tested by analysing HSP70 production in tachyzoites of the virulent RH strain of T. gondii recovered from mice deficient in: (1) T cells (nude mice); (2) T and B cells (SCID mice); (3) interferon-γ receptors (interferon-γ receptor knockout mice); and (4) tumour necrosis factor receptors (tumour necrosis factor receptor knockout mice). Parasites from nude and SCID mice produced as much HSP70 as immunocompetent mice. Likewise, T. gondii tachyzoites from mice lacking receptors for interferon-γ or tumour necrosis factor produced HSP70 in quantities similar to wild-type mice. The ability to produce reactive nitrogen intermediates in response to T. gondii infection, as detected by elevated levels of nitrate and nitrite in sera, was normal in tumour necrosis factor receptor knockout mice but was completely lacking in interferon-γ receptor knockout mice, indicating that reactive nitrogen intermediates are also not responsible for induction of parasite HSP70. Thus, immune factors that induce HSP70 production in mammalian cells do not appear to play primary roles in inducing HSP70 production by T. gondii.     

Activation of human neutrophils by Mycobacterium tuberculosis H37Ra involves phospholipase C gamma 2, Shc adapter protein, and p38 mitogen-activated protein kinase

Recent studies have shown that human neutrophils play a significant protective role in mycobacteria infection. When encountered with mycobacteria, neutrophils exhibit the typical early bactericidal responses including phagocytosis and generation of reactive oxygen intermediates (ROI), but the underlying mechanisms are largely unknown. The present study shows that stimulation of neutrophils with an attenuated strain of Mycobacterium tuberculosis H37Ra (Mtb) led to a tyrosine kinase-dependent ROI production in these cells. Stimulation with Mtb induces a rapid and transient tyrosine phosphorylation of several proteins, one of which was identified as phospholipase C gamma 2 (PLC gamma 2). Several tyrosine-phosphorylated proteins were associated with the PLC gamma 2 precipitates from Mtb-stimulated neutrophils, of which pp46 was characterized as the Shc adapter protein. A role for PLC gamma 2-Shc association in the generation of ROI is supported by the observations that stimulation with Mtb causes the activation of p38 mitogen-activated protein kinase (MAPK), a downstream target of the Shc/Ras signaling cascade, and that the effect of genistein on ROI production coincided with its ability to inhibit both PLC gamma 2-Shc association and p38 MAPK activation. Moreover, pretreatment of neutrophils with a PLC inhibitor markedly suppresses the Mtb-stimulated ROI production as well as p38 MAPK activation in these cells. Taken together, these results indicate that stimulation of neutrophils with Mtb triggers the tyrosine phosphorylation of PLC gamma 2 and its association with Shc, and that such association is critical for the Mtb-stimulated ROI production through activating p38 MAPK.     

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C). In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.     

5'-Adenosinephosphosulphate reductase (CysH) protects Mycobacterium tuberculosis against free radicals during chronic infection phase in mice

A major obstacle to tuberculosis (TB) control is the problem of chronic TB infection (CTBI). Here we report that 5'-adenosinephosphosulphate reductase (CysH), an enzyme essential for the production of reduced-sulphur-containing metabolites, is critical for Mycobacterium tuberculosis (Mtb) survival in chronic infection phase in mice. Disruption of cysH rendered Mtb auxotrophic for cysteine and methionine, and attenuated virulence in BALB/c and C57BL/6 immunocompetent mice. The mutant and wild-type Mtb replicated similarly during the acute phase of infection, but the mutant showed reduced viability during the persistent phase of the infection. The cysH mutant caused disease and death after 4-7 weeks of infection in four different groups of mice - Rag1(-/-), NOS2(-/-), gp91phox(-/-) NOS2(-/-) and gp91phox(-/-) mice given aminoguanidine [to suppress the effects of nitric oxide synthase 2 (NOS2)]- indicating minimal metabolic effect on the cysH mutant survival in these mice. The cysH mutant was also susceptible to peroxynitrite and hydrogen peroxide in vitro. These results show that CysH is important for Mtb protection during the chronic infection phase, and that resistance to nitrosative and oxidative stress may be the mechanism of this protection. Thus, this metabolic gene of an intracellular pathogen could have a secondary role in protection against the host immune response. Finally the lack of an endogenous human orthologue of cysH and its possible role in defence against adaptive immunity renders CysH an attractive enzyme for further studies as a target for therapeutics active against CTBI.     

Selenoprotein P protects cells from lipid hydroperoxides generated by 15-LOX-1

Reactive lipid hydroperoxides formed by lipoxygenases and cyclooxygenases can contribute to disease through cellular oxidative damage. Several selenoproteins have lipid hydroperoxidase activity, including glutathione peroxidase 4, thioredoxin reductase, and selenoprotein P (SelP). SelP is an extracellular glycoprotein that functions both in selenium distribution and has an antioxidant activity. The major objective of this study was to determine if an SelP, at physiological concentrations and in selenium replete media, possessed hydroperoxidase activity directed at lipid hydroperoxides generated from the metabolism of arachidonic acid by 15-lipoxygenase-1 (15-LOX-1). An SelP displayed in vitro lipid hydroperoxidase activity of 15-hydroperoxyeicosatetraenoic acid (15-HpETE), attenuated 15-HpETE oxidation in cellular assays, and in transcellular assay when 15-LOX-1 is metabolically active. These results suggest that an SelP can function as an antioxidant enzyme against reactive lipid intermediates formed during inflammation, but an SelP has modest activity. Nevertheless, this effect may help protect cells against the oxidative damage induced by these lipid metabolites.     

Exon ligation is proofread by the DExD/H-box ATPase Prp22p

To produce messenger RNA, the spliceosome excises introns from precursor (pre)-mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3' splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase.     

Comparative profiles of intramacrophage behavior of Mycobacterium tuberculosis and Mycobacterium avium complex with different levels of virulence

Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) strains with different levels of virulence in mice were examined for profiles of interaction with murine peritoneal macrophages (Mphis). Their growth rates in Mphis were in these orders: H37Ra strain (attenuated) > H37Rv strain (virulent) for MTB, and N-260 strain (moderate virulence) > MAC N-444 strain (low virulence) for MAC. MTB but not MAC caused the necrotic death of host Mphis in terms of increased release of lactate dehydrogenase from infected Mphis. The MTB H37Ra strain induced a greater production of reactive nitrogen intermediates (RNI) by Mphis than the MTB H37Rv strain did. However, this phenomenon was not observed with MAC, implying less important roles of RNI in the expression of Mphi antimicrobial activity against MAC organisms.     

Nitrosative stress in Escherichia coli: reduction of nitric oxide

The ability of enteric bacteria to protect themselves against reactive nitrogen species generated by their own metabolism, or as part of the innate immune response, is critical to their survival. One important defence mechanism is their ability to reduce NO (nitric oxide) to harmless products. The highest rates of NO reduction by Escherichia coli K-12 were detected after anaerobic growth in the presence of nitrate. Four proteins have been implicated as catalysts of NO reduction: the cytoplasmic sirohaem-containing nitrite reductase, NirB; the periplasmic cytochrome c nitrite reductase, NrfA; the flavorubredoxin NorV and its associated oxidoreductase, NorW; and the flavohaemoglobin, Hmp. Single mutants defective in any one of these proteins and even the mutant defective in all four proteins reduced NO at the same rate as the parent. Clearly, therefore, there are mechanisms of NO reduction by enteric bacteria that remain to be characterized. Far from being minor pathways, the currently unknown pathways are adequate to sustain almost optimal rates of NO reduction, and hence potentially provide significant protection against nitrosative stress.